Method of stabilizing aqueous pyrazoloacridone derivative solution

ABSTRACT

An aqueous solution of a pyrazoloacridone compound or pharmaceutical salt is stabilized using an acid, placing the solution in a well-closed container, evacuating air in the container, and sealing the container.

This application is a 371 of PCT/JP99/05643, filed Oct. 13, 1999.

TECHNICAL FIELD

The present invention relates to a method for stabilizing aqueoussolutions containing a pyrazoloacridone derivative or a pharmaceuticallyacceptable salt thereof, and well-closed containers containing theaqueous solution.

BACKGROUND ART

Antioxidants are used to prevent drugs from oxidative decomposition.However, it is known that the antioxidants cannot be added to somedrugs, since they would react with active ingredients or other additivesin preparations (J. Pharm. Sci., 61, 708 (1972)).

It is known that pyrazoloacridone derivatives have a DNA intercalationactivity and exhibit an antitumor effect (J. Med. Chem., 37, 1028(1994)). Specific examples of such pyrazoloacridone derivatives aredisclosed in Japanese Published Unexamined Patent Application No.1064/93.

Pyrazoloacridone derivatives or pharmaceutically acceptable saltsthereof are liable to decompose due to oxidation in aqueous solutions.Thus, there have been required stable aqueous solution preparationscontaining a pyrazoloacridone derivative or a pharmaceuticallyacceptable salt thereof which can be stored over a long period of time.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method forstabilizing aqueous solutions containing a pyrazoloacridone derivativeor a pharmaceutically acceptable salt thereof, and well-closedcontainers containing the aqueous solution.

The present invention relates to a method for stabilizing aqueoussolutions containing a pyrazoloacridone derivative or a pharmaceuticallyacceptable salt thereof, comprising adding an acid to an aqueoussolution containing a pyrazoloacridone derivative represented by thefollowing formula (I) (hereinafter referred to as Compound (I)):

wherein R^(1a), R^(1b), R^(1c) and R^(1d) independently representhydrogen, a lower alkyl group, —(CH₂)_(p)—X (wherein p is an integer of1 to 6; and X represents a hydroxyl group, a lower alkoxy group, or—NR^(2a)R^(2b) (wherein R^(2a) and R^(2b) independently representhydrogen, a lower alkyl group, —(CH₂)_(m)—Y (wherein m is an integer of1 to 6; and Y represents a hydroxyl group, a lower alkoxy group, or—NR^(3a)R³b (wherein R^(3a), and R^(3b) independently represent hydrogenor a lower alkyl group)), or R^(2a) and R^(2b) form a heterocyclic grouptogether with the nitrogen atom adjacent thereto)), or —CH((CH₂)_(n)OH)₂(wherein n is an integer of 1 to 5) or a pharmaceutically acceptablesalt thereof; substituting the air in a well-closed container containingthe aqueous solution with an inert gas; and sealing the container.

The present invention further relates to well-closed containerscontaining an aqueous solution containing Compound (I) or apharmaceutically acceptable salt thereof and an acid, wherein the air inthe well-closed container is substituted with an inert gas.

The lower alkyl group and the alkyl moiety in the lower alkoxy group inthe definition of formula (I) include linear or branched alkyl groupshaving from 1 to 6 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl sec-butyl, tert-butyl, pentyl, hexyl, and thelike. The heterocyclic group formed together with the adjacent nitrogenatom includes pyrrolidinyl, piperidino, piperazinyl, morpholino,thiomorpholino, quinolyl, pyrimidinyl, pyridazinyl, pyridyl, pyrrolyl,imidazolyl, pyrazolyl, and the like. Among these, pyrrolidinyl,piperidino, piperazinyl and morpholino are preferred.

Examples of the pharmaceutically acceptable salt of Compound (I) includeinorganic acid salts, such as hydrochlorides, hydrobromides, sulfates,phosphates, and the like, and organic acid salts, such as acetates,oxalates, malonates, maleates, fumarates, tartrates, succinates,citrates, and the like.

Compounds (I) are known compounds, which can be produced by, forexample, the production method described in Japanese PublishedUnexamined Patent Application No. 1064/93.

The concentration of Compound (I) in the aqueous solution is preferablyfrom 0.1 to 1,000 mM, more preferably from 1 to 100 mM, and particularlypreferably from 10 to 50 mM.

Examples of Compound (I) are shown in Table 1.

TABLE 1 (I)

Compound No. NR^(1a)R^(1b) NR^(1c)R^(1d) 1 NH(CH₂)₂NH₂ NH(CH₂)₂NH₂ 2N(C₂H₅)₂ NH(CH₂)₂NH₂ 3 N(C₂H₅)₂ NH(CH₂)₂N(CH₃)₂ 4 NH(CH₂)₂OH NH(CH₂)₂NH₂5 NH(CH₂)₂OH NH(CH₂)₃NH₂ 6 NH(CH₂)₂OH NH(CH₂)₂NH(CH₂)₂OH 7 NH(CH₂)₂OHNH(CH₂)₂NHCH₃ 8 NH(CH₂)₂OH NH(CH₂)₂N(CH₃)₂ 9 N[(CH₂)₂OH]₂NH(CH₂)₂N(CH₃)₂ 10 NHCH(CH₂OH)₂ NH(CH₂)₂NH₂ 11 NH(CH₂)₂OCH₃ NH(CH₂)₂NH₂12 NHCH(CH₂OH)₂ NH(CH₂)₂NH(CH₂)₂OH 13 NHCH(CH₂OH)₂ NH(CH₂)₃NH₂ 14NHCH(CH₂OH)₂

Examples of the acid include inorganic acids, organic acids, andinorganic salts thereof.

Examples of the inorganic acid include phosphoric acid, hydrochloricacid, sulfuric acid, nitric acid, and the like.

Examples of the organic acid include organic acids represented by thefollowing formula (II) (hereinafter referred to as Compound (II)):

R⁴R⁵CH—COOH  (II)

wherein R⁴ represents hydrogen or hydroxy; and R⁵ represents hydrogen,carboxy, or alkyl having from 1 to 3 carbon atoms which may besubstituted with hydroxy or carboxy. Examples of the alkyl having from 1to 3 carbon atoms in the definition of formula (II) include methyl,ethyl, propyl, isopropyl, and the like. The substitution number of thehydroxy or carboxy is 1 or 2. Examples of Compound (II) include lacticacid, glyceric acid, tartronic acid, malic acid, tartaric acid, and thelike. Lactic acid is particularly preferred as the organic acid.

Examples of the inorganic acid salt include alkali metal salts, such aslithium salts, sodium salts, potassium salts, and the like; and alkalineearth metal salts, such as beryllium salts, magnesium salts, calciumsalts, and the like.

The concentration of the acid in the aqueous solution is preferably from1 to 1,000 mM, more preferably from 5 to 500 mM, and particularlypreferably from 10 to 200 mM.

The material and shape of the well-closed container is not particularlylimited, so long as it can prevent the permeation of oxygen. Examples ofsuch material include glass, metals, resins, and the like. Examples ofthe resin include polyethylene, polystyrene, polycarbonate,polypropylene, polyvinyl chloride, 6-nylon, polyethylene terephthalate,and the like, with a resin having a small coefficient of oxygenpermeation being preferred. Examples of the resin having a smallcoefficient of oxygen permeation include resins having a coefficient ofoxygen permeation less than 0.1×10⁻¹¹ cm³ (STP) cm⁻¹s⁻¹cmHg⁻¹, such aspolyvinyl alcohol, polyacrylonitrile, polyvinylidene chloride, and thelike. Examples of such shape of the container include an ampul, a vial,a syringe, and the like.

The pH of the aqueous solution is from 1 to 7, preferably from 2 to 6,and particularly preferably from 3 to 5. The pH can be adjusted using analkali, such as sodium hydroxide, potassium hydroxide, or the like, oran inorganic acid, such as hydrochloric acid, sulfuric acid, or thelike.

The aqueous solution can contain a pharmaceutically acceptableantioxidant, solubilizing agent, isotonizing agent, surfactant, soothingagent, and the like, if desired. Examples of the antioxidant includeascorbic acid, vitamin E, L-cysteine, and the like. Examples of thesolubilizing agent include polyethylene glycol and the like. Examples ofthe isotonizing agent include glycerine, glucose, sodium chloride, andthe like. Examples of the surfactant include HCO-60 (manufactured byNikko Chemicals Co., Ltd.), and the like. Examples of the soothing agentinclude benzyl alcohol, lidocaine, and the like.

Examples of the inert gas include a nitrogen gas, an argon gas, a heliumgas, carbon dioxide, and the like. Among these, a nitrogen gas ispreferred.

The substitution of the air in the well-closed container with the inertgas can be carried out by a conventional method. For example, the inertgas may be poured after the well-closed container is depressurized bydrawing off the air therein with a vacuum pump or the like.Alternatively, the aqueous solution can be poured into the container inan inert gas atmosphere. Thus, the expression “substituting the air in awell-closed container containing the aqueous solution with an inert gas”as used in the present specification includes methods wherein theaqueous solution is poured into the container in an inert gasatmosphere, followed by sealing, and the expression “the air in thewell-closed container is substituted with an inert gas” includesconditions wherein the aqueous solution is poured into the container inthe inert gas atmosphere, followed by sealing. When the air in thecontainer is substituted with the inert gas, it is preferred that theinert gas concentration in the gas in the container is increased to 90%(v/v) or more. In the present invention, the inert gas concentration inthe gas in the container is more preferably 95% (v/v) or more, andparticularly preferably 99% (v/v) or more. The concentration of theinert gas in the gas in the container can be determined by directlymeasuring the inert gas concentration by a known method or by measuringthe oxygen gas concentration. The concentration of oxygen in the gas canbe measured, for example, using a trace oxygen analyzer RO-102-SP(manufactured by Iijima Electronics Corporation).

Hereinafter, Examples, Comparative Examples and Test Examples of thepresent invention are shown. However, the present invention is notlimited to these Examples. A concentration (%) of nitrogen is shown byv/v.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1

In water for injection, 50 mg of Compound 5 and 9 mg of lactic acid weredissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Using a freeze-dryer, the freeze-drycontainer was depressurized, and then a nitrogen gas was poured tosubstitute the air in the space of each vial with a nitrogen gas. Afterpouring the nitrogen gas, the vials were sealed with rubber stoppers andaluminum caps to produce an aqueous solution preparation of Compound 5.When analyzed with a trace oxygen analyzer RO-102-SP (manufactured byIijima Electronics Corporation), the nitrogen concentration in the gasin the space of the vial was 99.5%.

Example 2

In water for injection, 50 mg of Compound 5 was dissolved. Afteradjusting the pH to 4 by adding hydrochloric acid thereto, the totalvolume was made up to 10 mL. The solution was aseptically filteredthrough a sterile filter (0.22 μm, Acrodisc manufactured by GelmanScience), and a 1 mL portion each of the solution was poured in glassvials. Using a freeze-dryer, the freeze-dry container was depressurized,and then a nitrogen gas was poured to substitute the air in the space ofeach vial with a nitrogen gas. After pouring the nitrogen gas, the vialswere sealed with rubber stoppers and aluminum caps to produce an aqueoussolution preparation of Compound 5. When analyzed with a trace oxygenanalyzer RO-102-SP (manufactured by Iijima Electronics Corporation), thenitrogen concentration in the gas in the space of the vial was 99.5%.

Example 3

In water for injection, 50 mg of Compound 5 and 9 mg of lactic acid weredissolved. After adjusting the pH to 6 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Using a freeze-dryer, the freeze-drycontainer was depressurized, and then a nitrogen gas was poured tosubstitute the air in the space of each vial with a nitrogen gas. Afterpouring the nitrogen gas, the vials were sealed with rubber stoppers andaluminum caps to produce an aqueous solution preparation of Compound 5.When analyzed with a trace oxygen analyzer RO-102-SP (manufactured byIijima Electronics Corporation), the nitrogen concentration in the gasin the space of the vial was 99.5%.

Example 4

In water for injection, 50 mg of Compound 5 and 6 mg of acetic acid weredissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Using a freeze-dryer, the freeze-drycontainer was depressurized, and then a nitrogen gas was poured tosubstitute the air in the space of each vial with a nitrogen gas. Afterpouring the nitrogen gas, the vials were sealed with rubber stoppers andaluminum caps to produce an aqueous solution preparation of Compound 5.When analyzed with a trace oxygen analyzer RO-102-SP (manufactured byIijima Electronics Corporation), the nitrogen concentration in the gasin the space of the vial was 99.5%.

Example 5

In water for injection, 50 mg of compound 5 and 13.6 mg of potassiumdihydrogenphosphate were dissolved. After adjusting the pH to 4 byadding hydrochloric acid thereto, the total volume was made up to 10 mL.The solution was aseptically filtered through a sterile filter (0.22 μm,Acrodisc manufactured by Gelman Science), and a 1 mL portion each of thesolution was poured in glass vials. Using a freeze-dryer, the freeze-drycontainer was depressurized, and then a nitrogen gas was poured tosubstitute the air in the space of each vial with a nitrogen gas. Afterpouring the nitrogen gas, the vials were sealed with rubber stoppers andaluminum caps to produce an aqueous solution preparation of Compound 5.When analyzed with a trace oxygen analyzer RO-102-SP (manufactured byIijima Electronics Corporation), the nitrogen concentration in the gasin the space of the vial was 99.5%.

Example 6

In water for injection, 50 mg of Compound 5 and 15 mg of tartaric acidwere dissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Using a freeze-dryer, the freeze-drycontainer was depressurized, and then a nitrogen gas was poured tosubstitute the air in the space of each vial with a nitrogen gas. Afterpouring the nitrogen gas, the vials were sealed with rubber stoppers andaluminum caps to produce an aqueous solution preparation of Compound 5.When analyzed with a trace oxygen analyzer RO-102-SP (manufactured byIijima Electronics Corporation), the nitrogen concentration in the gasin the space of the vial was 99.5%.

Comparative Example 1

In water for injection, 50 mg of Compound 5 and 9 mg of lactic acid weredissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Then, the vials were sealed with rubberstoppers and aluminum caps to produce an aqueous solution preparation ofCompound 5.

Comparative Example 2

In water for injection, 50 mg of Compound 5 was dissolved. Afteradjusting the pH to 4 by adding hydrochloric acid thereto, the totalvolume was made up to 10 mL. The solution was aseptically filteredthrough a sterile filter (0.22 μm, Acrodisc manufactured by GelmanScience), and a 1 mL portion each of the solution was poured in glassvials. Then, the vials were sealed with rubber stoppers and aluminumcaps to produce an aqueous solution preparation of Compound 5.

Comparative Example 3

In water for injection, 50 mg of Compound 5 and 9 mg of lactic acid weredissolved. After adjusting the pH to 6 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Then, the vials were sealed with rubberstoppers and aluminum caps to produce an aqueous solution preparation ofCompound 5.

Comparative Example 4

In water for injection, 50 mg of Compound 5 and 6 mg of acetic acid weredissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Then, the vials were sealed with rubberstoppers and aluminum caps to produce an aqueous solution preparation ofCompound 5.

Comparative Example 5

In water for injection, 50 mg of Compound 5 and 21 mg of citric acidwere dissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Then, the vials were sealed with rubberstoppers and aluminum caps to produce an aqueous solution preparation ofCompound 5.

Comparative Example 6

In water for injection, 50 mg of Compound 5 and 13.6 mg of potassiumdihydrogenphosphate were dissolved. After adjusting the pH to 4 byadding hydrochloric acid thereto, the total volume was made up to 10 mL.The solution was aseptically filtered through a sterile filter (0.22 μm,Acrodisc manufactured by Gelman Science), and a 1 mL portion each of thesolution was poured in glass vials. Then, the vials were sealed withrubber stoppers and aluminum caps to produce an aqueous solutionpreparation of Compound 5.

Comparative Example 7

In water for injection, 50 mg of Compound 5 and 15 mg of tartaric acidwere dissolved. After adjusting the pH to 4 by adding sodium hydroxidethereto, the total volume was made up to 10 mL. The solution wasaseptically filtered through a sterile filter (0.22 μm, Acrodiscmanufactured by Gelman Science), and a 1 mL portion each of the solutionwas poured in glass vials. Then, the vials were sealed with rubberstoppers and aluminum caps to produce an aqueous solution preparation ofCompound 5.

Test Example 1

The aqueous solution preparations produced in the above Examples 1 to 6and Comparative Examples 1 to 7 were stored in a thermostat at 60° C.for 60 days. The residual ratio of Compound 5 was measured by highperformance liquid chromatography.

Analytical conditions for high performance liquid chromatography:

Column: YMC-Pack ODS-AM AM-312

Mobile phase: 0.1 M phosphate buffer (pH 3.0) containing 10 mM1-octanesulfonic acid:acetonitrile (73:27, w/w)

Flow rate: 1.5 mL/min

Detection wavelength: 267 nm

The results are shown in Table 2.

TABLE 2 Residual ratio of Compound 5 (%) Example 1 91.7 Example 2 81.5Example 3 73.3 Example 4 89.4 Example 5 88.3 Example 6 92.7 ComparativeExample 1 26.9 Comparative Example 2 38.9 Comparative Example 3 16.2Comparative Example 4 30.5 Comparative Example 5 16.0 ComparativeExample 6 36.8 Comparative Example 7 21.8

Test Example 2

The aqueous solution preparations produced in the above Examples 1 to 6and Comparative Examples 1 to 7 were stored in a thermostat at 60° C.for 60 days. Then the coloration and dissolution conditions of eachaqueous solution were observed with the naked eye.

The results are shown in Table 3.

TABLE 3 Change in Dissolution coloration condition Example 1 − ClearExample 2 ++ Clear Example 3 ++ Clear Example 4 + Clear Example 5 +Clear Example 6 + Slightly cloudy Comparative Example 1 +++ ClearComparative Example 2 +++ Clear Comparative Example 3 +++ ClearComparative Example 4 +++ Clear Comparative Example 5 +++ Oily and verycloudy Comparative Example 6 +++ Clear Comparative Example 7 +++ Oilyand very cloudy Score of change in coloration −: No change. +: Slightincrease in coloration is observed. ++: Noticeable increase incoloration. +++: Highly noticeable increase in coloration.

INDUSTRIAL APPLICABILITY

According to the present invention, aqueous solution preparationscontaining a pyrazoloacridone derivative or a pharmaceuticallyacceptable salt thereof can be stored at stable conditions over a longperiod of time.

What is claimed is:
 1. A method for stabilizing an aqueous solutioncontaining a pyrazoloacridone compound or a pharmaceutically acceptablesalt thereof, comprising: adding an acid to a container of an aqueoussolution comprising a pyrazoloacridone compound represented by formula(I):

 wherein R^(1a), R^(1b), R^(1c) and R^(1d) independently representhydrogen, a lower alkyl group, —(CH₂)_(p)—X (wherein p is an integer of1 to 6; and X represents a hydroxyl group, a lower alkoxy group, or—NR^(2a)R^(2b) (wherein R^(2a) and R^(2b) independently representhydrogen, a lower alkyl group, —(CH₂)_(m)—Y (wherein m is an integer of1 to 6; and Y represents a hydroxyl group, a lower alkoxy group, or—NR^(3a)R^(3b) (wherein R^(3a) and R^(3b) independently representhydrogen or a lower alkyl group)), or R^(2a) and R^(2b) form aheterocyclic group together with the nitrogen atom adjacent thereto)),or —CH((CH₂)_(n)OH)₂ (wherein n is an integer of 1 to 5) or apharmaceutically acceptable salt thereof; evacuating air from thecontainer; and sealing the container.
 2. The method as claimed in claim1, wherein the acid is an organic acid represented by the followingformula (II): R⁴R⁵CH—COOH  (II) wherein R⁴ represents hydrogen orhydroxy; and R⁵ represents hydrogen, carboxy, or alkyl having from 1 to3 carbon atoms which may be substituted with hydroxy or carboxy.
 3. Themethod as claimed in claim 1, wherein the acid is lactic acid.
 4. Themethod as claimed in any of claims 1 to 3, wherein the aqueous solutionhas a pH of 2 to
 6. 5. A well-closed container comprising an aqueoussolution containing a pyrazoloacridone compound represented by formula(I):

wherein R^(1a), R^(1b), R^(1c) and R^(1d) independently representhydrogen, a lower alkyl group, —(CH₂)_(p)—X (wherein p is an integer of1 to 6; and X represents a hydroxyl group, a lower alkoxy group, or—NR^(2a)R^(2b) (wherein R^(2a) and R^(2b) independently representhydrogen, a lower alkyl group, —(CH₂)_(m)—Y (wherein m is an integer of1 to 6; and Y represents a hydroxyl group, a lower alkoxy group, or—NR^(3a)R^(3b) (wherein R^(3a) and R^(3b) independently representhydrogen or a lower alkyl group)), or R^(2a) and R^(2b) form aheterocyclic group together with the nitrogen atom adjacent thereto)),or —CH((CH₂)_(n)OH)₂ (wherein n is an integer of 1 to 5), or apharmaceutically acceptable salt thereof, and an acid, wherein anatmosphere in the well-closed container does not contain air.
 6. Thewell-closed container as claimed in claim 5 wherein said acid is anorganic acid represented by the following formula (II):R⁴R⁵CH—COOH  (II) wherein R⁴ represents hydrogen or hydroxy; and R⁵represents hydrogen, carboxy, or alkyl having from 1 to 3 carbon atomswhich may be substituted with hydroxy or carboxy.
 7. The well-closedcontainer as claimed in claim 5, wherein the acid is lactic acid.
 8. Thewell known container as claimed in any of claims 5 to 7, wherein theaqueous solution has a pH of 2 to
 6. 9. The method as claimed in any ofclaims 1 to 3, wherein the evacuated container is filled with an inertgas.
 10. The method as claimed in claim 9, wherein the inert gas isnitrogen gas.
 11. The method as claimed in claim 4, wherein theevacuated container is filled with an inert gas.
 12. The well-closedcontainer as claimed in any of claims 5-7, wherein the atmosphere insidethe container is inert.
 13. The well-closed container as claimed inclaim 8, wherein the atmosphere inside the container is inert.
 14. Thewell-closed container as claimed in claim 12, wherein the inert gas isnitrogen gas.
 15. The well-closed container as claimed in claim 13,wherein the inert gas is nitrogen gas.
 16. The method as claimed inclaim 11, wherein the inert gas is nitrogen gas.